Abstract

Visualizing Structural Variations Of Single DNA Molecules In A Nanofluidic Device

Rodolphe Marie, Assistant Professor, Technical University of Denmark

Single DNA molecules can be studied using nanofluidics. The base pair sequence and the methylation pattern of DNA have been characterized from images of fluorescently labeled single molecules stretched in nanochannels. In nanochannels the stretching depends strongly on the buffer composition and the sub-micrometer geometry of the device. We designed a nanofluidic device based on a cross-shaped nanoslit. Long DNA fragments (>440 microns) were imaged at 98% stretching. The high stretching minimizes the number of base pairs within the diffraction limit and thus provides the highest possible resolution achievable with conventional epifluorescence microscopy (about 1 kilobase). The device requires no sub-micron lithography and allows stretching DNA at a wide range of buffer conditions. We imaged 1.4 mega base pair long denaturation-renaturation (DR) maps created from fluorescently labeled fragments of human DNA. DR maps provide a fingerprint of the underlying base pair sequence. The high-resolution DR maps enabled us (i) to map each fragment to its origin in the human reference genome, and (ii) to detect large-scale structural variations (2-5kb up to 0.5 Mb) in single molecules. That is, high-resolution DR maps can be used to characterize structural variations in single copies of the human genome.